Identification, validation and biological characterisation of novel glioblastoma tumour microenvironment subtypes: implications for precision immunotherapy.

BACKGROUND: New precision medicine therapies are urgently required for glioblastoma (GBM). However, to date, efforts to subtype patients based on molecular profiles have failed to direct treatment strategies. We hypothesised that interrogation of the GBM tumour microenvironment (TME) and identification of novel TME-specific subtypes could inform new precision immunotherapy treatment strategies. MATERIALS AND METHODS: A refined and validated microenvironment cell population (MCP) counter method was applied to >800 GBM patient tumours (GBM-MCP-counter). Specifically, partition around medoids (PAM) clustering of GBM-MCP-counter scores in the GLIOTRAIN discovery cohort identified three novel patient clusters, uniquely characterised by TME composition, functional orientation markers and immune checkpoint proteins. Validation was carried out in three independent GBM-RNA-seq datasets. Neoantigen, mutational and gene ontology analysis identified mutations and uniquely altered pathways across subtypes. The longitudinal Glioma Longitudinal AnalySiS (GLASS) cohort and three immunotherapy clinical trial cohorts [treatment with neoadjuvant/adjuvant anti-programmed cell death protein 1 (PD-1) or PSVRIPO] were further interrogated to assess subtype alterations between primary and recurrent tumours and to assess the utility of TME classifiers as immunotherapy biomarkers. RESULTS: TMEHigh tumours (30%) displayed elevated lymphocyte, myeloid cell immune checkpoint, programmed cell death protein 1 (PD-1) and cytotoxic T-lymphocyte-associated protein 4 transcripts. TMEHigh/mesenchymal+ patients featured tertiary lymphoid structures. TMEMed (46%) tumours were enriched for endothelial cell gene expression profiles and displayed heterogeneous immune populations. TMELow (24%) tumours were manifest as an 'immune-desert' group. TME subtype transitions upon recurrence were identified in the longitudinal GLASS cohort. Assessment of GBM immunotherapy trial datasets revealed that TMEHigh patients receiving neoadjuvant anti-PD-1 had significantly increased overall survival (P = 0.04). Moreover, TMEHigh patients treated with adjuvant anti-PD-1 or oncolytic virus (PVSRIPO) showed a trend towards improved survival. CONCLUSIONS: We have established a novel TME-based classification system for application in intracranial malignancies. TME subtypes represent canonical 'termini a quo' (starting points) to support an improved precision immunotherapy treatment approach.

Adherence ability and serum resistance of different hospital clusters of Acinetobacter baumannii.

The role of mechanical ventilation and catheters in favouring Acinetobacter baumannii infections needs to be better understood. This study evaluated the adherence of 19 isolates of different hospital clusters of A. baumannii to abiotic surfaces and epithelial cells (HEp-2). Of the hydrophobic isolates, 80% adhered to polystyrene, indicating a close relationship between hydrophobicity and adherence. All isolates adhered to epithelial cells to different degrees, and 73·7% showed an aggregated pattern. Analysis of the serum resistance of catheter-tip isolates showed that all were resistant. These worrisome results showed that the high capacity of A. baumannii to adhere to surfaces and survive in human serum could hinder treatment and control of this pathogen.

Experimental results and related clinical implications of PET detection of epidermal growth factor receptor (EGFr) in cancer.

The epidermal growth factor receptor (EGFr) is one of the most studied molecules as a target for cancer therapy. Over these last few years, several studies attempting to identify predictive biomarkers of treatment response, such as the receptor status or other molecules related to the downstream signalling pathway, have been conducted. However, from a clinical point of view, the information obtained from ex vivo analyses still has various limitations that may be overcome by the combination with molecular imaging technologies which may provide a noninvasive, global, in vivo evaluation of the molecular tumour background. The aim of this review is to report the preclinical results of all positron emission tomography (PET) tracers synthesized until now for in vivo detection of EGFr in cancer. Two classes of PET compounds have been developed: labelled small molecules such as tyrosine kinase inhibitors and labelled monoclonal antibodies. The in vitro and in vivo results of these PET tracers are very different depending on the chemical properties, positron emission radionuclide, or animal models. As a consequence, various critical questions are still open, and the implications of a translation in the clinical setting for EGFr imaging in cancer patients is discussed.

Molecular imaging and targeted therapies in oncology: new concepts in treatment response assessment. a collection of cases.

The widespread use of several new non-cytotoxic drugs and the significant improvements in functional imaging highlights a number of difficulties in monitoring, interpreting and predicting treatment response in clinical practice. Certain guidelines for disease assessment after therapy are already available: the traditional Response Evaluation Criteria in Solid Tumours guidelines based on tumour size variations using conventional imaging technologies, the recent combined method developed by Choi and colleagues in gastrointestinal stromal tumour treated with tyrosine kinase inhibitors based on tumour density variations using computed tomography (CT), and the European Organization for Research and Treatment of Cancer criteria based on tumour glucose metabolism variations using fluorodeoxyglucose (FDG) positron emission tomography (PET). At the moment combined PET/CT response criteria are still not available. A number of new PET compounds other than FDG are also currently being developed to visualize specific cellular and molecular tumour pathways but their role in assessment and prediction of cancer treatment response has not yet been thoroughly investigated in a large series. However, in clinical practice many oncologists treat cancer patients with targeted therapies or chemotherapy and evaluate the response using conventional or functional imaging without appropriate and standardized guidelines. The aim of this study was to present a selection of clinical cases that illustrate the usefulness of new PET tracers and efficacy evaluation of new drugs. In the era of molecular imaging and molecular therapies, these cases highlight the urgency to develop new criteria for treatment assessment and the exigency of correctly interpreting the biological information obtained from new technologies, and introduce new concepts that require further investigation in clinical trials.

Efficacy of glutamine-supplemented parenteral nutrition on short-term survival following allo-SCT: a randomized study.

Fifty-three patients with hematological malignancies who underwent Allo-SCT from HLA-identical siblings were randomly assigned to receive glutamine-enriched parenteral nutrition-PN (GlPN, n=27) or standard PN (PN, n=26), in isonitrogenous solutions. Deaths (D+100 and D+180), infections, acute GVHD, length of stay, time of neutropenia and intestinal permeability (IP) were studied. Ages, gender, diagnosis, disease status and treatment variables were equally distributed between groups. Survival on D+180 was increased in GlPN (74%) vs PN (46%), P=0.03 (log-rank), as on D+100 (P=0.05). Most deaths occurred before D+100, especially in PN (10/26, 39%) vs GlPN (4/27, 15%). GVHD was the most frequent cause of death (8/21, 38%), especially in PN (n=6, five before D+100). Other outcomes were not affected. IP was affected on admission, was not affected by glutamine enrichment, but consistently worsened throughout the study. Results showed that GlPN was efficacious in increasing short-term survival after Allo-SCT. Benefits of glutamine seem to be independent of mucosal protection, as IP was not affected by its use. A trend to a lower incidence of GVHD deaths may suggest an immunomodulatory role of glutamine.

Radiolabelling, quality control and radiochemical purity assessment of the Octreotide analogue 68Ga DOTA NOC.

Somatostatin receptors 1-5 are over expressed in neuroendocrine tumours (NETs). 68Ga-labelled [1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid]-1-Nal3-Octreotide (DOTA NOC), a recent synthesized somatostatin analogue, shows high affinity for those receptors. Herein, modifications of a commercial module for the labelling of DOTA NOC with 68Ga, as well as the assessment of time course of the radiochemical purity variation are described. The evaluation of radiochemical stability was done by two different chromatographic methods: reversed-phase radio HPLC and fast TLC analysis. Labelled compound has been found radiochemically stable within 3h from the end of labelling (EOL) and radiochemical purity was always higher than 99%. After 73 labelling sessions the system showed great reproducibility and high radiochemical yield.

Interpretation of 11C-choline PET/CT for the diagnosis of local relapse in radically treated prostate cancer.

PURPOSE: 11C-choline PET/CT is a widely-used tool for the diagnostic of prostate cancer (PCa). In literature, a great variability of local relapse (LR) detection rate is reported. The aim of this study is to provide positivity criteria for 11C-choline PET/CT detection of LR in patients who had surgery for PCa and presented prostate specific antigen (PSA) failure. METHODS: Sixty patients radically treated for PCa and presenting PSA failure were retrospectively analysed. Two Nuclear Medicine Physicians revised the 11C-choline PET/CT scans and defined by consensus if even mild focal uptake was present in the prostate bed (PB) and bladder-urethral junction (BUJ) along midline, regardless the previous report results.The results were subsequently correlated with a clinical and radiological follow up (FU) of 1 to 2 year and with TNM staging, Gleason score (GS), PSA level at relapse, radiotherapy (RT) and hormone therapy (HT) after surgery. RESULTS: There was focal uptake in 22/60 patients; 11 of them were true positive and 11 false positive. The PSA level showed a tight connection with the true positivity/negativity of Choline scan. Most of true positive cases (10/11 patients) presented a PSA ≥ 1 ng/ml, while approximately half of the false positive cases (5/11 patients) presented PSA below 1 ng/ml. The other variables were not correlated to Choline detection rate for LR. CONCLUSIONS: This study shows that an even mild focal uptake of Choline in the PB and BUJ along midline must be considered suspicious for LR in patients radically treated for PCa, especially if they are presenting with PSA level > 1 ng/ml.

Subcutaneous fentanyl infusion in a patient with bowel obstruction and renal failure.

Inoperable bowel obstruction in patients with renal failure is a difficult clinical situation. In the last days of life, an accumulation of morphine metabolites in patients with impaired renal function may cause opioid toxicity, including terminal agitation. The use of an alternative drug may prevent morphine metabolite accumulation in uremic patients. Fentanyl may be an alternative to morphine. It has a large apparent volume of distribution, a short plasma half-life, and extensive biotransformation without active metabolites. A patient with acute renal impairment and bowel obstruction was successfully treated with a subcutaneous continuous infusion of fentanyl (25 micrograms/hr) and boluses of 12.5 micrograms for the last 2 days of life, limiting the worsening of the dramatic clinical picture of bowel obstruction combined with renal impairment. No local toxicity was evidenced.

Long-term ketamine subcutaneous continuous infusion in neuropathic cancer pain.

Neuropathic cancer pain may be less responsive to opioids than other pain. Several studies suggest that N-methyl-D-aspartate (NMDA)-receptor antagonists could play a role in the treatment of neuropathic pain. Ketamine is an NMDA-receptor antagonist that is used as an anesthetic and has been suggested as a useful drug for neuropathic pain. Subanesthetic doses of ketamine can yield analgesia without hypnosis. We describe a patient who developed neuropathic cancer pain unresponsive to opioid escalation and spinal administration of a combination of bupivacaine-morphine and was subsequently treated by subcutaneous continuous ketamine infusion. A starting dose of 150 mg/day provided good pain relief and a dramatic reduction of the oral morphine dose (from 5 g to 200 mg). A slow and progressive increase of ketamine and morphine dosage (400 mg and 200 mg by the subcutaneous route, respectively) continued to provide adequate pain relief after 13 months of therapy despite signs of progressive disease.

Use of the retention index in gas chromatographic studies of drugs.

In earlier studies, it has been shown that the retention index (Ir) is a very reproducible measure of gas chromatographic mobility. Statistically comparing Ir values obtained here with those found in references has yielded very satisfying results. This will enable the use of Ir values in the screening phase of analysis for general unknowns using the great number of data offered by references; thus limiting the range of inquiry to no more than 6 or 7 drugs, provided that the unknown is between the scheduled ones. An index of analytical behavior that is an absolutely reproducible number will be necessary for computerizing toxicological analysis.

Nutritional value of a rice-hydrolysate formula in infants with cows' milk protein allergy: a randomized pilot study.

This study was designed to assess whether a rice-hydrolysate formula allows normal growth and adequate metabolic balance in infants with cows' milk protein allergy. Infants (seven females, nine males; aged 6-14 months) were randomly assigned to receive a rice-hydrolysate formula (n = 8) or a soy formula (control group, n = 8). Standardized growth indices (Z scores) and biochemical parameters were evaluated during a 6-month treatment period. Infants in both groups showed normal growth patterns during the study, and no adverse reactions were seen. Mean plasma biochemical parameters were within the normal ranges, and did not differ between groups. In conclusion, rice-hydrolysate formula may be a nutritionally suitable alternative for infants with cows' milk protein allergy. Larger studies, with satisfactory power, should be undertaken to confirm these findings.

Clinical and pro-host effects of cefaclor in prophylaxis of recurrent otitis media in HIV-infected children.

The aim of this study was to evaluate the efficacy of cefaclor in the prophylaxis of recurrent acute otitis media (AOM) in human immunodeficiency virus (HIV)-infected children. The study was carried out in children born between 1 January 1986 and 31 December 1996 who had been vertically HIV infected Patients who had experienced recurrent AOM between October 1997 and March 1998 (period 1) were eligible for the trial. Recurrent AOM was defined as the occurrence in the same patient of three or more episodes of AOM within 6 months of the observation period. Patients recruited for this trial received cefaclor at a dose of 20 mg/kg once daily for 6 months between April and September 1998 (period 2). Clinical observation was carried out in periods 1 and 2 and for the first 6 months after prophylaxis, i.e. October 1998 - March 1999 (period 3). Natural killer-cell activity, phagocytosis and myeloperoxidase activity were determined before and at the end of the prophylactic period. For each period, CD4-cell count measurement and CD4-positive cell class were recorded. Seventeen children were recruited for this trial. No significant differences were observed in natural killer-cell activity between periods 1 and 2, nor were any significant differences observed in CD4-positive cell class or CD4-positive cell count between the three periods. However, cefaclor administration was associated with a reduction in the number of AOM episodes in 100% of cases and a mean increase in myeloperoxidase activity in 57% of cases. This suggests that cefaclor may be useful in the prophylaxis of recurrent AOM in HIV-infected children.

[Use of RIA (radioimmunoassay) technics in the identification of narcotics in biological fluids]. / Applicazione delle tecniche "R.I.A." alla identificazione di sostanze stupefacenti in liquidi biologici

Radioimmunoassay has been compared with extraction thin-layer chromatography and gas-chromatography for the detection of morphine and barbiturate in urine. Radioimmunoassay has been found to be the most sensitive and rapid method and its use in large scale screening and forensic toxicology is considered.

The detection of morphine and codeine in human teeth: an aid in the identification and study of human skeletal remains.

When studying unidentified putrefied or skeletonised human remains it may be difficult to obtain information on drug habits which may prove important for the construction of a biological profile or lead to hypotheses on the manner of death. The detection of morphine and codeine in teeth from human remains may prove crucial in obtaining such information and thus give forensic odontology and anthropology a further tool for identification. Because teeth can be an important deposit of exogenous substances accumulated both in the pulp and in the calcified tissues, they are an invaluable source of data from a toxicological point of view. The authors therefore tested 3 groups of teeth for morphine and codeine: the first group consisted of artificially aged teeth from individuals known to have died of heroin overdose; the second, of teeth from individuals with no history of drug abuse; the third, of teeth from cases of burnt, putrefied and skeletonised remains found in conditions strongly suggestive of a drug-related death. Results showed that in groups 1 and 3 morphine and codeine could still be identified in the teeth, proving that these tissues may be a reliable source for toxicological information concerning the history of the individual. Further studies are needed to verify whether the substances detected reflect drugs in circulation in an acute phase (and therefore present in blood vessels in the pulp) or whether they represent drugs which have percolated and been stored in dentine and enamel and thus denote a history of drug abuse. Nonetheless this study shows that teeth may be an important source of toxicological information in the forensic scenario.

[Chemical-toxicological diagnosis in 16 cases of "intravenous narcotism" deaths (author's transl)]. / La diagnosi chimico-tossicologica nelle morti da morfinici: considerazioni su 16 casi.

The chemical-toxicological diagnosis aspects involved in 16 cases of "intravenous narcotism" deaths are discussed. Liquids and biological materials were directly tested by RIA, GLC and TLC. According to the Authors, RIA is assumed to be more speedy and more sensitive than traditional chemical technics. Therefore RIA is proposed for quantitative determination of morfina and its derivates in biological liquids and viscera, previous the qualitative confirmation by the chemical tests.

Monte Carlo modeling provides accurate calibration factors for radionuclide activity meters.

Accurate determination of calibration factors for radionuclide activity meters is crucial for quantitative studies and in the optimization step of radiation protection, as these detectors are widespread in radiopharmacy and nuclear medicine facilities. In this work we developed the Monte Carlo model of a widely used activity meter, using the Geant4 simulation toolkit. More precisely the "PENELOPE" EM physics models were employed. The model was validated by means of several certified sources, traceable to primary activity standards, and other sources locally standardized with spectrometry measurements, plus other experimental tests. Great care was taken in order to accurately reproduce the geometrical details of the gas chamber and the activity sources, each of which is different in shape and enclosed in a unique container. Both relative calibration factors and ionization current obtained with simulations were compared against experimental measurements; further tests were carried out, such as the comparison of the relative response of the chamber for a source placed at different positions. The results showed a satisfactory level of accuracy in the energy range of interest, with the discrepancies lower than 4% for all the tested parameters. This shows that an accurate Monte Carlo modeling of this type of detector is feasible using the low-energy physics models embedded in Geant4. The obtained Monte Carlo model establishes a powerful tool for first instance determination of new calibration factors for non-standard radionuclides, for custom containers, when a reference source is not available. Moreover, the model provides an experimental setup for further research and optimization with regards to materials and geometrical details of the measuring setup, such as the ionization chamber itself or the containers configuration.

Automation synthesis modules review.

The introduction of (68)Ga labelled tracers has changed the diagnostic approach to neuroendocrine tumours and the availability of a reliable, long-lived (68)Ge/(68)Ga generator has been at the bases of the development of (68)Ga radiopharmacy. The huge increase in clinical demand, the impact of regulatory issues and a careful radioprotection of the operators have boosted for extensive automation of the production process. The development of automated systems for (68)Ga radiochemistry, different engineering and software strategies and post-processing of the eluate were discussed along with impact of automation with regulations.

Use of 65Zn as a tracer for the assessment of purification in the 68Ga-DOTANOC synthesis.

In the last years (68)Ga has got into the focus of researchers and clinicians especially for radio-labeling of biomolecules; an important characteristic of this positron emitting isotope is its availability via the (68)Ge/(68)Ga generator system: the long-lived (68)Ge (t1/2=270.8 d) produces the short-lived (68)Ga (t1/2=67.63 min) which decays to stable (68)Zn. (68)Ge breakthrough compromises (68)Ga radionuclidic purity, while (68)Zn might affect the specific activity of the radiopharmaceutical. In this paper we investigated the weight of these impurities in (68)Ga-DOTANOC synthesis. (65)Zn (t1/2=244.26d; decay mode: EC 98.3%, ß(+) 1.7%) was used as a radiotracer of stable (68)Zn; samples of the purification columns, wastes and product were recovered and measured with a calibrated HPGe gamma-ray spectrometry system. The results showed that (68)Zn competes with (68)Ga in labeling DOTANOC with a (95±2)% labeling yield; they also proved the effectiveness of the STRATA X-C cationic post-processing of the generator eluate in lowering the amount of this impurity to less than 1%. Moreover this approach, along with the purification of the final product through a STRATA X cartridge, effectively removes (68)Ge breakthrough providing a (68)Ga-DOTANOC radionuclidic purity of (99.9999986±0.0000006)%, superior to 99.9% required by the Pharmacopoeia Monograph on (68)Ga Edotreotide injection.

Automated fast procedure for the simultaneous extraction of hair sample performed with an automated workstation.

INTRODUCTION: Hair testing has a leading role in toxicology practice and even more in those aspects tightly linked to the assessment of psychoactive drug use and abuse in social life. AIM: The objective of the present study was to develop and validate an automated SPE sample-preparation step, suited for GC/MS confirmation analysis of basic drugs in hair drug control. The method was studied and optimized for quantitative determination and in a second time it was extended to real hair samples. The purpose of method validation was to ensure good reliability, reproducibility and quickness. METHODS: Janus Automated Workstation (PerkinElmer) was employed to perform SPE hair extraction, using 96-well plate SPEC MP1 acquired from Varian (Agilent Technologies). After derivatization of dried extracts, screening confirmations were performed using gas chromatography (GC) followed by mass spectrometry (MS). GC/MS data were validated following standard guidelines, but our attention was focused on three headings: samples cross-contamination, "memory effect" and extraction recovery. RESULTS: Validation requests were fully accomplished and we always obtained best results with the automated procedure. For instance, analytes mean recovery was between 70 and 90% and data analysis proved that no contamination between samples occurred. CONCLUSIONS: The automated workstation has shown good reliability (cross contamination and "memory effect" were tested and excluded), effectiveness (no false negative was detected), solvent saving (500µL/sample vs traditionally LLE 4mL/sample) and quickness (50min for 96 tests cycle).